Silica-based nanoparticles (NPs) pose great potential for medical and biological applications; however their interactions with living cells have not been investigated in full. of phalloidin fluorescence dropped within the same line: Control – Si – SiB. It could be suggested that the effects of silica-based particles may result in structural reorganization of cortical cytoskeleton with subsequent stiffness increase and concomitant F-actin content decrease (for example in recruitment of additional actin-binding proteins within membrane and regrouping of actin filaments). within a short period of time using AFM. In view of the above the main objective of this study was to determine the mechanical characteristics of mesenchymal stem cells when cultured in the presence of silica and silica-boron nanoparticles. Methods Isolation of mesenchymal stem cells and their cultivation conditions In order to obtain the primary culture a method of enzymatic processing of the stromal vascular fraction isolation from human lipoaspirates was used [17 18 The obtained cells were cultivated in α-MEM medium (MP Biomedicals Santa Ana CA USA) with 2 mM of glutamine (PanEco Moscow Russia) 100 IU/mL of penicillin 100 μ/mL of streptomycin (PanEco) and 10% fetal bovine serum (Hyclone Logan UT USA) added to the culture. The cell seeding density was 3?×?103 cells/cm2. Standard cultivation was performed at 37°C and under 5% CO2 using a CO2 cultivator (Sanyo Moriguchi Osaka Japan). The cells of passages 3 to 5 5 were used for the experiments. Silica (Si) and silica-boron (SiB) NPs were added to the culture medium at the same concentration of 100 μg/mL. Cultivations were performed for 1 and 24 h. Nanoparticles were prepared at the Prokhorov General Physics Institute RAS by the method described in detail previously . Evaluation of mesenchymal stem cell viability RO5126766 The proportion of AnV?+?cells (early apoptosis) AnV+/PI?+?cells (post-apoptotic necrosis) and PI?+?cells (necrosis) was determined using an Annexin V-FITC/PI kit (Beckman Coulter Brea CA USA) and Epic XL flow cytofluorimeter (Beckman Coulter) in strict accordance with the standard procedure stated in the manufacturer’s manual. At least 10 0 events were analyzed. Atomic force microscopy Atomic force microscopy (AFM) is a useful tool for studying cell mechanics [20 21 Measurements of transversal stiffness in this study were conducted using a Solver P47-Pro instrument (NT-MDT Moscow Russia) in accordance with a RO5126766 technique which has previously been described in detail . For each cantilever the stiffness (N/m) was adjusted using the resonance position. When working in liquid soft cantilevers were used with the stiffness coefficient of approximately 0.01 N/m. The contact mode was applied to record the Rabbit Polyclonal to IL18R. force curves. The radius of curvature ((m/A). Then the force curves were recorded on cells obtaining the ratio is the measured cantilever deviation (A) and is the generalized indentation depth (m). Further the actual indentation depth and the force applied to it were calculated using the following formulae: h s ?=?x?-?y?·?a F x ?=?y?·?a?·?kc where hc is the actual indentation depth (m) F x is the actual force applied to a cell (N) and kc is the cantilever stiffness coefficient. Finally at the indentation depth of 60 nm the change of applied force was determined and RO5126766 the stiffness of a sample was estimated using the following formula: ks?=?F x /hs. The obtained results were processed using MATLAB 6.5 software which was specially developed for this research. Confocal microscopy Structures of fibrillar actin (F-actin) were detected using standard TRITC-phalloidin (Sigma St. Louis MO USA) staining. Cells that had previously been washed off the medium were fixed with 4% paraformaldehyde solution for 15 min. In order to RO5126766 permeabilize the cells 0.1% Triton X-100 (Sigma) detergent was added to the prefixed cells for 15 min. Then the cells were rinsed twice with phosphate-buffered saline (PBS). Further TRITC-phalloidin was added to the cells at a concentration of 50 μg/mL and cultured at 37°C for 40 min. Then the cells were rinsed thrice with PBS. In order to maintain the fluorescence the samples were covered by the specific water-soluble Fluoroshield medium containing DAPI (Sigma) to achieve.